Pseudomonas aeruginosa is a gram negative opportunistic pathogen which causes serious infections in severely burned patients, immuno-compromised hosts, and Cystic Fibrosis patients. Toxin A is one of the most toxic virulence factors produced by P. aeruginosa. The synthesis of toxin A in P. aeruginosa is highly regulated by different environmental factors especially iron. Despite several studies, the exact mechanism of this regulation is not completely understood. The long term goal of this proposal is to determine the mechanisms of regulating toxin A synthesis in P. aeruginosa and the factors involved in this regulation. Recently, a new toxA positive regulatory gene, ptxR, was isolated. Analysis of ptxR showed that it enhances transcription of both toxA and the previously described toxA regulatory gene, regAB. Nucleotide sequence analysis revealed the presence of an open reading frame which codes for a 34 kDa predicted protein. Computer analysis revealed the presence of an adjacent gene which interferes with ptxR function. An open reading frame (ORF2) which is divergently transcribed from ptxR (from the minus strand) and codes for a 30 kDa protein was identified. This predicted protein has a significant homology to several proteins of the GalR family of repressors. The specific aims of this proposal are: 1) to examine the mechanism of ptxR function; 2) to determine the transcriptional regulation of ptxR; and 3) to examine the effect of ORF2 on ptxR function. Examining the mechanism of ptxR function will include the purification of ptxR production, DNA/protein binding experiments (gel retardation experiments), phosphorylation experiments, two dimensional gel experiments, and the construction of a PAO1 isogenic mutant in ptxR. Regulatory studies will include transcriptional analysis of ptxR, regA, and toxA. Analysis of ORF2 function will involved in vivo and in vitro transcription experiments, co-immunoprecipitation experiments, and the isolation of an ORF2 isogenic mutant.